Mice models of human prostate cancer progression

ABSTRACT

The present invention provides an immune deficient mouse having a human prostate xenograft of locally advanced or metastatic prostate cancer and uses thereof.

This invention was made with Government support under Grant No.GM-08243, awarded by the National Institute of Health. The Governmenthas certain rights in this invention.

This application is a continuation-in-part of U.S. Ser. No. 08/732,676,filed Oct. 15, 1996, now abandoned the contents of which is herebyincorporated by reference in its entirety.

Throughout this application, various publications are referenced withinparentheses. Full citations of these publications may be found at theend of the specification immediately preceding the claims. Thedisclosures of these publications are hereby incorporated by referenceherein in their entireties.

BACKGROUND OF THE INVENTION

Prostate cancer is the most common cause of cancer in men. In 1996,317,000 new cases of prostate adenocarcinoma were diagnosed and over41,400 men died of the disease (Karp et al., 1996). Only lung cancer hasa higher mortality. The chance of a man developing invasive prostatecancer during his lifetime is 1 in 6 or 15.4%. At the age of 50, a manhas a 42% chance of developing prostate cancer and 2.9% of dying fromthe disease. While advances in early diagnosis and treatment of locallyconfined tumors have been achieved, prostate cancer is incurable once ithas metastasized. Patients with metastatic prostate cancer on hormonaltherapy will eventually develop an androgen-refractory (androgenindependent) state that will lead to disease progression and death.

The major cause of morbidity and mortality from prostate cancer is theresult of androgen-independent metastatic tumor growth. As a result,there is great interest in defining the molecular basis for advancedstaged disease with the hope that these insights may improve thetherapeutic options for these patients. However, progress in this areahas been difficult for a number of reasons. For example, theavailability of prostate tissue for molecular studies is limited becausemost prostate tumors are small. Moreover, there is tremendousheterogeneity within surgical prostatectomy tumor samples, it isdifficult to reducibly culture prostate cancer explants in vitro, andthere are a limited number of immortalized prostate cancer cell lines.

There is, therefore, an interest in finding alternative procedures whichwill allow for stable growth of prostate cancer tissue, which in turnwould allow for the investigation of the progression of prostate cancerin vivo, provide a stable supply of prostate cancer tissue and provide amodel for metastatic expansion of prostate cancer which accuratelysimulates or mimics the biology of the disease.

There is also a need for more reliable and informative staging andprognostic methods in the management of advanced prostate cancer.Clinically staging prostate tumors relies on rectal examination todetermine whether the tumor remains within the borders of the prostaticcapsule (locally confined) or extends beyond it (locally advanced), incombination with serum PSA determinations and transrectal ultrasoundguided biopsies. However, none of these techniques has proven reliablefor predicting progression of the disease.

The primary sites of prostate cancer metastasis are the regional lymphnodes and bone. Bone metastases occur in sites of hematopoieticallyactive red bone marrow, including lumbar vertebral column, ribs, pelvis,proximal long bones, sternum and skull. Bony metastases of prostatecancer differ from those of other tumors that commonly colonize in bonein that they are characterized by a net gain in bone formation(osteoblastic) rather than resorption predominant in bone metastases ofbreast cancer and melanoma.

Until recently, bone metastasis was thought to be a late stage indisease progression. However, the recent development of highly sensitivetechniques (such as RT-PCR for prostate specific genes) to detectprostate cancer cells has revised this notion. Prostate cancer cellshave been detected in the peripheral blood and bone marrow of patientswith advanced stage disease using RT-PCR assays for PSA mRNA (Ghosseinet al., 1995; Seiden et al., 1994; Wood et al., 1994; Katz et al., 1994)or immunomagnetic bead selection for PSA protein (Brandt et al., 1996).When positive, these tests show that prostate cancer cells representabout 0.1-1.0% of the circulating blood cells. Moreover, it is now clearthat small numbers of prostate cancer cells circulate in the peripheralblood and lodge in the bone marrow even in patients with early stage,low risk disease (Olsson et al., 1997; Deguchi et al., 1997; Katz etal., 1996). Interestingly, these cells tend to disappear in mostpatients following radical prostatectomy (Melchior et al., 1997). Theseresults suggest that the primary tumor site is a constant source forseeding the marrow, and that only a small subset of these cells have thecapacity to grow into a metastatic lesion. This concept is consistentwith estimates from animal models for other tumor types that only about1 in 10,000 circulating cancer cells are able to lodge in andproductively colonize other organs (Fidler et al., 1990).

The factors involved in advanced prostate cancer progression to bonemetastasis are poorly defined. Anatomic, local bone/marrow and tumorcell factors are all believed to play a role. Baston described theextensive vertebral venous system that consists of a network oflongitudinal, valveless veins that run parallel to the vertebral columnand form extensive, direct anastomoses with the veins of the ribs,pelvis and brain (Baston, 1942). Prostate cancer cells enteringprostatic veins may be transported via this plexus directly to theseorgans without entering the inferior vena cava of passing through thelungs. This hypothesized mechanism of metastasis both by clinicaldocumentation of patterns of prostate cancer metastasis compared toother tumors and by animal models wherein occlusion of inferior venacava during tail vein injection of tumor cells increased the incidenceof vertebral metastasis (Nishijima et al., 1992; Coman and DeLong,1951).

Although the vascular anatomy is an essential component of the spread ofprostate cancer to bone, it cannot fully explain the selective patternof all skeletal metastases. Bone, which receives 5-10% of the cardiacoutput, is a more frequent metastatic site than would be expected fromblood-flow criteria (Berettoni and Carter, 1986). Bone marrow consistsof two clearly identifiable components: the hematopoetic cells whichcomprise the majority of the cellular elements, and stromal componentthat is formed of highly vascular connective tissue. The hematopoeticcells are transient in the bone marrow; upon maturation they move intothe blood stream. The stroma, however, remains and serves as ascaffolding upon which the hematopoetic cells can differentiate andmature. One of the important factors in prostate cancer cells arrestingin these sites is likely their adhesion to the bone marrow stroma. Ithas been demonstrated both in vitro and in vivo that tumor cells willpreferentially adhere to the stromal cells of the organs to which theymetastasize (Haq et al., 1992; Netland and Zetter, 1985; Zetter et al.,1992). When rat prostate cancer (MatLyLu) cells were injected into theleft ventricle of syngenic rats, vertebral body metastases developed;these metastases were then collected, disaggregated and reinjected. Celllines established after 6 similar passages through animals adheredstrongly and preferentially to bone marrow stroma and endothelial cells(Haq et al, 1992). A similar approach has increased the incidence ofmetastasis from the LNCaP prostate cancer cell line in immune deficientmice (Thalmann et al., 1994).

It is critical that appropriate in vivo models for prostate cancer bonemetastasis be developed to more fully explore the mechanistic aspects ofthis process. To date, most work in this area has focused on three humanprostate cancer cell lines--PC-3, DU-145, and LNCaP (Lee et al., 1993).All three grow a subcutaneous nodules in immune deficient mice, andsublines with variable metastatic properties have been derived (Shervinet al., 1988, 1989; Wang and Sterarns, 1991; Kozlowski et al., 1988).However, none of these sublines has been shown to reproducibly give riseto osteoblastic lesions typical of prostate cancer. A major limitationof the DU-145 and PC-3 cell lines is the lack of prostate specificantigen (PSA) and androgen receptor (AR) expression (Kaighn et al.,1979; Gleave et al., 1992), which raises regarding relevance to clinicalprostate cancer. The LNCaP cell line is androgen responsive andexpresses PSA, but contains a mutation in the androgen receptor whichalters ligand specificity.

SUMMARY OF THE INVENTION

The invention provides animal xenograft models of human prostate cancerprogression capable of simulating or mimicking the development ofprimary tumors, micrometastasis, and the formation of osteoblasticlesions characteristic of late stage disease. The model may be used tostudy the stagewise progression of prostate cancer. In this regard, theinvention replicates the process of cell migration from the primarytumor site to distant sites of micrometastasis, including bone marrow,as well as the development of macrometastatic osteoblastic bone lesionsfrom micrometastatic precursors. The models are also capable ofduplicating the clinical transition from androgen dependent to androgenindependent tumor growth characteristic of advanced prostate cancerpatients undergoing androgen ablation therapy. In addition, methods forpropagating prostate cancer cells within the various stages of prostatecancer as well as methods for isolating and expanding stage-specificprostate cancer cell populations are also provided. Further, theinvention provides a unique, serially-passaged, androgen-sensitiveprostate cancer cell line which expresses PSA, wild-type androgenreceptor, and prostate acid phosphatase. The models and methods of theinvention provide a system for studying the molecular biology ofprostate cancer, evaluating the influence that various genes andtherapeutic compounds have on distinct stages of disease progression,assessing the metastatic potential of prostate cancer cells, anddesigning patient-specific therapeutic regimens.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one color drawing executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee.

FIGS. 1A-B. Molecular analysis of prostate cancer xenografts for humanDNA content and expression of prostate specific antigen (PSA). FIG. 1A:DNA-PCR analysis of genomic DNA isolated from xenografts using primersspecific for the human β-globin gene. Each sample shown was obtainedfrom late passage xenografts. The LAPC-5 sample was obtained at passage4 when the human tumor was overgrown by a tumor of murine origin. Thetwo LAPC-4 samples were obtained from androgen dependent ("ad") andandrogen independent ("ai") sublines FIG. 1B: RT-PCR analysis of totalRNA using primers specific for human PSA and human or murine β-actin ("%human cells" refers to the percent of LNCaP cells which are diluted into10⁵ mouse cells). A dilution series of human prostate cancer LNCaP cellsinto murine NIH 3T3 cells is shown on the left side of the figure, withthe percentage of LNCaP cells varying from 100% to 0.0%. The resultsfrom the three LAPC xenografts are shown on the right.

FIG. 2. Photographs of immunohistochemical analysis of the LAPC-4xenograft, showing expression of PSA. Paraffin sections offormalin-fixed tissue from the original tumor sample obtained at thetime of surgery (top row) and the LAPC-4 xenograft (bottom row) werestained with hematoxylin and eosin (left), a control antibody (middle)and an antibody specific for human PSA (right).

FIG. 3. Bar graphs showing androgen sensitivity of the LAPC-3 and LAPC-4xenografts in vivo. Equal size implants of the LAPC-3 and LAPC-4xenografts were passaged simultaneously into male or female mice andexamined weekly for the formation of tumors.

FIGS. 4A-B. Regression and regrowth of LAPC-4 tumors followingcastration. FIG. 4A: Line graph showing typical results from two animalsin the cohort whose tumor sizes were equivalent at 4 weeks. The timecourse for tumor development in a female mouse is shown for comparison.FIG. 4B: Bar graph showing the average tumor size (+/- standard error)from the entire cohort of intact and castrated male mice. The data fromeach animal are expressed as tumor size relative to the 4 week timepoint.

FIG. 5. Line graph showing limiting dilution analysis of LAPC-4engraftment in male mice.

FIGS. 6A-B. Photographs showing detection of micrometstatic disease inmice bearing LAPC-4 xenografts. Total RNA was isolated from the murinetissues indicated and analyzed for the expression of PSA (a) or β-actin(b) using RT-PCR. The results from the tumor and various tissues ofthree representative mice are shown. Tissues from a fourth mouse(control SCID) were analyzed as a negative control. The signal can bequantified by comparison to with LNCaP (lane 1). No RNA was added to thenegative control sample (lane 2).

FIG. 7. Photographs showing detection of LAPC-4 cells in bone byimmunohistochemistry after 2 weeks. Frozen sections of the tibia of miceinjected with LAPC-4 cells were stained with an antibody tocytokeratin-18 (bottom panel) or an isotype control antibody (toppanel). The four cells staining red are LAPC-4 cells.

FIGS. 8A-C. Photographs showing LAPC-4 causes bone lesions. Hematoxylinand eosin sections of the tibia are shown at 4, 6 and 8 weeks followingintratibial injection of LAPC-4 cells. Panel A shows a small focus oftumor formation adjacent to normal bone and hematopoiesis. Panels B andC show progressive increase in new bone formation in response tosurrounding tumor cells.

FIG. 9. Radiographic evidence of osteoblastic bone lesions induced byLAPC-4. X-rays of mice were performed at 8 weeks post injection ofLAPC-4 cells in the tibia (right panel). The bone shows evidence oferosion of the cortex with enhanced bone density in the marrow cavitydue to osteoblastic activation.

FIG. 10. Flow cytometry analysis of LAPC-4 cells stained withanti-galectin-6 antibody, showing the expression level relative to anisotype control antibody.

DETAILED DESCRIPTION OF THE INVENTION

Immune Deficient Animal Hosts

Severe combined immune deficient (SCID) mice are the preferred animalhost utilized in the practice of the invention. Various other immunedeficient mice, rodents or animals may be used, including those whichare deficient as a result of a genetic defect, which may be naturallyoccurring or induced, such as, for example, nude mice, Rag 1 and/or Rag2 mice, and the like, and mice which have been cross-bred with thesemice and have an immunocompromised background. The deficiency may be,for example, as a result of a genetic defect in recombination, agenetically defective thymus or a defective T-cell receptor region.Induced immune deficiency may be as a result of administration of animmunosuppressant, e.g. cyclosporin, removal of the thymus, etc. Varioustransgenic immune deficient mice are currently available or can bedeveloped in accordance with conventional techniques. Ideally, theimmune deficient mouse will have a defect which inhibits maturation oflymphocytes, particularly lacking the ability to rearrange the T-cellreceptor region. Female, male, castrated or uncastrated mice may beemployed, depending upon whether one is interested in studying theeffect of the availability of androgens on the course of the tumorgrowth. In the particular and preferred embodiments described herein,C.B. 17 scid/scid mice are used. In addition to mice, immune deficientrats or similar rodents may also be employed in the practice of theinvention.

Models that Simulate Advanced Prostate Cancer

One aspect of the invention provides murine xenograft models whichsimulate or mimic human prostate cancer from primary tumor formation.Also provided are methods for propagating advanced stage human prostatetumor tissue as subcutaneous xenografts in immune deficient mice. In thepractice of the invention, prostate cancer xenografts may be establishedin immune deficient mice by the subcutaneous implantation of fresh humanprostate cancer explants surgically removed from patients with locallyadvanced or metastatic prostate cancer. The site of implantation may beinto any subcutaneous site which will permit blood supply to reach theimplant, such as the flanks of the host animal. Tissue from primaryprostate tumors as well as from sites of lymph node, lung, bone, andother organ metastases may be used to establish the prostate cancerxenografts of the invention. Prostate tumor explants may be introducedin conjunction with a basement membrane composition, such as Matrigel(U.S. Pat. No. 5,508,188), an extracellular matrix preparation which hasbeen shown to enhance the growth of epithelial tumors in vivo (includingprostate cancer cells)(Lim et al., 1993; Noel et al., 1992; Pretlow etal., 1991), as well as other similar types of compositions. Onceestablished, the xenograft tumors grow to considerable size, providingsubstantial tissue volumes for further use. Xenografts of the inventionretain the human phenotype as determined by human β-globin expression,express human prostate specific antigen (PSA), and retain androgensensitivity and metastatic growth characteristics reflective of theclinical situation.

As used herein, the term "locally advanced prostate cancer" and "locallyadvanced disease" mean prostate cancers which have extended through theprostate capsule, and are meant to include stage C disease under theAmerican Urological Association (AUA) system, stage C1-C2 disease underthe Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM(tumor, node, metastasis) system. In general, surgery is not recommendedfor patients with locally advanced disease, and these patients havesubstantially less favorable outcomes compared to patients havingclinically localized (organ-confined) prostate cancer. Locally advanceddisease is clinically identified by palpable evidence of indurationbeyond the lateral border of the prostate, or asymmetry or indurationabove the prostate base. Locally advanced prostate cancer is diagnosedpathologically following radical prostatectomy if the tumor invades orpenetrates the prostatic capsule, extends into the surgical margin, orinvades the seminal vesicles.

As used herein, the terms "metastatic prostate cancer" and "metastaticdisease" mean prostate cancers which have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

This and other aspects of the invention described herein provide toolsfor studying the pathogenesis and treatment of advanced prostate cancer.For example, immune deficient mice bearing subcutaneous (and other)xenografts may be used to evaluate the effect of various prostate cancertreatments (e.g., therapeutic compositions, gene therapies,immunotherapies, etc.) on the growth of tumors and progression ofdisease. Xenograft cells may be used to identify novel genes and geneswhich are differentially expressed in prostate cancer cells, or toanalyze the effect such genes have on the progression of prostatecancer. For example, the genetic compositions of prostate cancer cellsfrom xenografts having differing androgen sensitivities (e.g., androgendependent vs. androgen independent) may be compared to each other aswell as to the genetic compositions of normal prostate cells. Likewise,the genetic compositions of micrometastatic prostate cancer cells may becompared to those of metastatic prostate cancer cells. Various nucleicacid subtraction and sampling techniques may be used for this purpose,including, for example, representational difference analysis (RDA). Inaddition, prostate cancer xenograft cells may be used for theintroduction of various genetic capabilities, including the introductionof various genes, antisense sequences, ribozymes, regulatory sequenceswhich enhance or repress the expression of endogenous genes, and soforth.

In addition, this aspect of the invention provides methods for purifyingprostate cancer cells from the heterogeneous mixture of cells typical ofhuman prostate cancer biopsy material, further providing methods forgenerating greater quantities of tumor cells for subsequent use andanalysis. In one embodiment, the method for purifying prostate cancercells comprises implanting human prostate cancer biopsy materialsubcutaneously into a SCID or other immune deficient mouse and allowingthe implanted material to grow as a xenograft in the mouse. The purifiedhuman prostate cancer cells are obtained by harvesting the xenograft.Xenografts may be expanded and further purified by serial propagation inadditional immune deficient mice or by propagation in short term cellculture. Single cell suspensions of xenograft tumor tissue or culturedcells may be used to orthotopically seed intraprostatic tumors, bonetumors or other organ tumors. Xenograft tumor tissue and cellpreparations may be frozen and viably recovered for later use.

The invention also provides subcutaneous prostate cancer xenograftswhich retain stable prostate cancer cell phenotypes through multiplepassages in SCID mice. Various embodiments are provided, includingandrogen dependent and androgen independent xenografts, xenografts whichexpress prostate specific antigen (PSA) at clinically reflective levels,xenografts which express wild-type androgen receptor (AR), andxenografts which exhibit chromosomal abnormalities. Still otherembodiments include xenografts which retain all of the foregoingcharacteristics as well as xenografts that model the progression toandrogen independent disease. These and other embodiments of theinvention are described in more detail by way of the examples whichfollow. As described in Example 1, a number of subcutaneous xenograftswere successfully established from tumor tissue explants taken from theprostate gland and from bone, lymph, and lung metastases of patientswith stage C or D prostate cancer. These xenografts grow and passage inSCID mice with high frequency and retain definitive characteristics ofhuman prostate cancer, even in late passages. One xenograft, designatedLAPC-4, has been adapted to tissue culture as a stable cell line and hasbeen in continuous culture for 18 months.

Xenografts such as the LAPC-4 (LAPC-4 cell line deposited with AmericanType Culture Collection, Manassas, Va., on Mar. 3, 2000, ATCC No. 1441)xenograft described in Example 1 are of particular interest. Similar toprostate tumors isolated directly from patients, LAPC-4 cells retainexpression of prostate specific antigen (PSA), androgen receptor (AR),and prostatic acid phosphatase through more than 20 passages. Moreover,the LAPC-4 xenograft is unique among prostate cancer model systems sinceits AR contains no mutations in the DNA or ligand binding domains and ARexpression is retained in androgen independent LAPC-4 sublines. Inaddition, the LAPC-4 xenograft models the transition fromandrogen-dependent to androgen-independent disease as well as thedevelopment of micrometastatic disease. For example, LAPC-4 tumorspassaged in male mice retain androgen-dependent growth characteristics,whereas tumors passaged in castrated males or female mice acquire astable androgen-independent phenotype. These sublines can be easilyexpanded using the methods of the invention to provide ample tissue formolecular and biochemical analysis of events associated withandrogen-independent growth. There are few other experimental models forandrogen-dependent prostate cancer growth. Published reports include thewidely used LNCaP cell line (Lim et al., 1993; Gleave et al., 1992) andtwo recently described xenografts, CWR22 (Weinstein et al., 1994) andLuCaP23 (Lin et al., 1996). The LAPC-4 xenograft is unique becausetumors placed under the selective pressure of androgen deprivationreproducibly evolve to an androgen-independent state, providing anopportunity to evaluate the molecular changes associated withandrogen-independence over time and directly test their functionalimportance.

This aspect of the invention also provides assays for determining thefunction or effect of various genes on prostate cancer cells. In oneembodiment, the assay comprises isolating prostate cancer cells from aprostate cancer xenograft (e.g., subcutaneous, intraprostatic),transducing the cells with the gene of interest such that the transducedcells express or overexpress the gene, establishing a subcutaneous orintraprostatic xenograft tumor in a SCID or other immune deficient mousewith the transduced cells, and evaluating the growth of the resultingxenograft. The effect of expressing the gene on the growth of thexenograft may be determined by reference to a control xenograftestablished with untransduced prostate cancer cells, preferably isolatedfrom the same parental xenograft. In another embodiment, the assaycomprises generating a prostate cancer xenograft (e.g., subcutaneous,intraprostatic), transducing the cells of the xenograft with the gene ofinterest in vivo, and evaluating the growth of the xenograft, whereinthe effect of the gene on the growth of the xenograft may be determinedby reference to a control xenograft.

Similarly, the invention provides assays for determining the effect ofcandidate therapeutic compositions or treatments on the growth ofprostate cancer cells. In one embodiment, the assay comprises applyingthe composition or treatment to a SCID or other immune deficient mousebearing a subcutaneous human prostate cancer xenograft and determiningthe effect of the treatment on the growth of the xenograft. In anotherembodiment, a SCID or other immune deficient mouse bearing anintraprostatic xenograft is used to determine the effect of thecomposition or treatment.

This aspect of the invention may also have various clinicalapplications, including using the model in a method to assess prognosisof a patient with locally advanced or metastatic prostate cancer. Forexample, in one embodiment, the method comprises implanting a prostatetumor sample from the patient into an immune deficient mousesubcutaneously, and allowing the implanted sample to grow as a xenograftin the mouse. The rates of xenograft growth may be used as a prognosticindicator. The results of such analysis may assist a treating oncologistin determining how aggressively to treat a patient.

Models that Simulate Prostate Cancer Micrometastasis

Another aspect of the invention provides models and methods forsimulating and studying the process of micrometastasis in human prostatecancer. SCID mice bearing subcutaneous prostate cancer xenografts showevidence of circulating prostate cancer cells. Thus, this modelduplicates the process of cell migration from the primary tumor to thebone marrow and other distant sites of micrometastasis. As detailed inExample 3, 100% of male mice inoculated subcutaneously with xenograftLAPC-4 cells developed localized subcutaneous tumors within 4 to 6 weekswithout evidence of bony metastasis. However, when these animals wereexamined for the presence of micrometastatic disease, up to 50% of themice had detectable prostate cancer cells in bone marrow and blood.Using the same semi-quantitative RT-PCR assay that has been applied tolarge surveys of prostate cancer patients, micrometastatic prostatecancer cells were found at levels comparable to about 0.1 to 1.0% of thetotal mouse bone marrow. Similar results were obtained byimmunohistochemical analysis for PSA expression. Thus, subcutaneousgrowth of prostate cancer xenografts mimics the clinical observationthat prostate cancer cells circulate in the blood and lodge in the bonemarrow, even in early stage disease.

In one embodiment, simulating or mimicking prostate cancermicrometastasis comprises establishing a subcutaneous prostate cancerxenograft in a SCID or other immune deficient mouse and allowing thetumor to grow for a time sufficient to permit the detection of prostatecancer cells in the peripheral blood of the mouse. The presence ofmicrometastasis is monitored by detecting prostate tumor cells whichhave migrated to the lympahtic and/or vascular system, bone, lung,liver, and/or other sites distant from the primary xenograft site.Detection of such cells may be accomplished by, for example, assayingfor the presence of human PSA mRNA in the peripheral blood using anRT-PCR assay for PSA mRNA (such as the assay described in Example 3).

In another embodiment, simulating prostate cancer micrometastasiscomprises preparing a single cell suspension of prostate cancer cellsfrom a subcutaneous xenograft tumor grown in a SCID (or other immunedeficient) mouse, followed by intraprostatic (orthotopic) injection ofthe single cell suspension into another SCID (or other immune deficient)mouse. The intraprostatic tumor is allowed to grow for a time sufficientto permit the detection of prostate cancer cells in the peripheral bloodof the mouse or in other sites distant from the orthotopic tumor. Singlecell suspensions prepared from cultured xenograft cells may also be usedfor intraprostatic (orthotopic) implantation.

This aspect of the invention also provides a framework for testing theeffect of certain variables on the development of micrometastasis. Suchvariables may include the presence or absence of hormones or othergrowth-modulating factors in the environment of the tumor, theexpression status of various genes within the tumor cells, etc. Forexample, the rate of micrometastasis of androgen dependent and androgenindependent xenograft variants may be evaluated. Such an evaluation isdescribed in Example 3, using the androgen dependent and independentsublines of the LAPC-4 xenograft, demonstrating a significantly higherrate of micrometastasis in mice bearing the androgen independent LAPC-4xenografts.

In this regard, the invention provides assays for determining thefunction or effect of various genes on the progression of prostatecancer micrometastasis. In one embodiment, the assay comprises isolatingprostate cancer cells from a prostate cancer xenograft (e.g.,subcutaneous, intraprostatic), transducing the cells with the gene ofinterest such that the transduced cells express or over-express thegene, using the transduced cells to establish a subcutaneous orintraprostatic xenograft tumor in a SCID or other immune deficientmouse, and evaluating the presence and levels of micrometastatic diseaseby detecting prostate cancer cells in blood, bone marrow, lymph nodes,and/or other sites distant from the site of the primary xenograft tumor.The effect of expressing the gene on the rate of micrometastasis may bedetermined by reference to a control xenograft established withuntransduced prostate cancer cells, referably isolated from the sameparental xenograft. In another embodiment, the assay comprisesgenerating a prostate cancer xenograft (e.g., subcutaneous,intraprostatic), transducing the cells of the xenograft with the gene ofinterest in vivo, and evaluating the presence and levels ofmicrometastatic disease, wherein the effect of expressing the gene onthe rate of micrometastasis may be determined by reference to a controlxenograft.

Similarly, the invention provides assays for determining the effect ofcandidate therapeutic compositions or treatments on the progression tomicrometastatic disease. In one embodiment, the assay comprises applyingthe composition or treatment to a SCID mouse bearing a subcutaneoushuman prostate cancer xenograft, and determining the effect of thetreatment on micrometastasis by monitoring the presence and levels ofprostate cancer cells in the peripheral blood, lymph nodes, bone marrow,and/or other sites distant from the xenograft. In another embodiment, aSCID or other immune deficient mouse bearing an intraprostatic xenograftis used to determine the effect of the treatment on micrometastasis.

This aspect of the invention may also have various clinicalapplications, including using the model in a method to assess prognosisof a patient with locally advanced or metastatic prostate cancer. Forexample, in one embodiment, the method comprises implanting a prostatetumor sample from the patient into an immunocompromised mousesubcutaneously and allowing the implanted sample to grow as a xenograftin the mouse. The rates of xenograft growth and the development ofmicrometastasis may be used as prognostic indicators. The results ofsuch analysis may assist a treating oncologist in determining howaggressively to treat a patient.

Models that Simulate Metastatic Prostate Cancer:

Another aspect of the invention provides models and methods formimicking and studying the development of macrometastatic osteoblasticbone lesions (bone metastasis) in prostate cancer. Subcutaneous growthof xenograft tumors results in detectable micrometastasis, indicatingthat cells from xenograft tumors in SCID mice have the ability to exitthe site of primary tumor growth, circulate in blood, and lodge in thebone marrow, reflecting the human clinical situation.

In one embodiment, simulating the development of prostate cancer bonemetastasis comprises injecting a single cell suspension of prostatecancer cells prepared from a subcutaneous prostate cancer xenograftgrowing in SCID (or other immune deficient) mouse into the prostate ofanother SCID (or other immune deficient) mouse host, and allowing theresulting orthotopic tumor to grow for a time sufficient to permit thedetection of bone metastasis in the mouse. Alternatively, subcutaneousprostate cancer xenografts may be established with such single cellpreparations and allowed to grow. Detection of bone metastasis may beaccomplished by various means, including histologically,immunohistochemically, and radiographically.

Subcutaneous and orthotopic tumors typically grow quickly, reaching asize which demands that the host animal be sacrificed within about 4-6weeks. Therefore, alternative methods which increase the number ofprostate cancer cells in the bone marrow, thereby obviating thislimitation, are also provided. In one embodiment, a single cellsuspension prepared from xenograft tumor cells, or from xenograft cellsin tissue culture, is injected directly into the bone marrow cavity(e.g., tibial) of a SCID or other immune deficient mouse. Thedevelopment of micrometastasis, bone tumor growth, and osteoblasticactivity may be monitored in various ways, including byimmuno-histochemistry and in situ hybridization of bone sections or byradiographic imaging.

As described in Example 6, a single cell suspension of 10,000 xenografttumor cells prepared from a subcutaneous tumor was injected into thetibia of a SCID mouse. A small subset of the injected cells wasdetectable at 2 weeks, followed by small foci of bone tumor growth in afew isolated areas at 4 weeks, followed by extensive macroscopic bonetumor growth, destruction of bone cortex, and net new bone formation by6-8 weeks. Accordingly, cells isolated from the human prostate cancerxenografts of the invention are capable of proliferation in themicroenvironment of the SCID mouse bone marrow cavity.

The foregoing method provides an excellent model for simulating theformation of osteoblastic bone lesions and the progression to this stageof the disease. The model may be used not only to study the molecularand cellular events involved in the progression of this stage ofprostate cancer, but also to test the effect of various candidatetherapeutic genes, proteins and other compounds. In addition, the modelmay be used as an assay for assessing the metastatic and osteoblasticpotential of prostate cancer cells obtained from human patients.

Accordingly, the invention also provides assays for determining thefunction or effect of various genes on the progression of prostatecancer bone metastasis. In one embodiment, the assay comprises isolatingprostate cancer cells from a prostate cancer xenograft (e.g.,subcutaneous, intraprostatic, bone), transducing the cells with the geneof interest such that the transduced cells express or over-express thegene, introducing the transduced cells into the bone marrow cavity of aSCID or other immune deficient mouse, and monitoring the bone marrow forthe presence and levels of osteoblastic macrometastatic lesions. Theeffect of expressing the gene on the development and growth of bonemetastasis may be determined by reference to a control animal receivinguntransduced prostate cancer cells, preferably isolated from the sameparental xenograft. In another embodiment, the assay comprisesgenerating a bone marrow xenograft in a SCID (or other immune deficient)mouse by injecting a single cell suspension of prostate cancer cellsprepared from a subcutaneous or intraprostatic xenograft established inanother SCID (or other immune deficient )mouse, transducing the cells ofthe bone marrow xenograft with the gene of interest in vivo, andevaluating the effect of the gene on the presence and levels ofosteoblastic macrometastatic lesions.

Further, the invention provides assays for determining the effect ofcandidate therapeutic compositions or treatments on the progression ofprostate cancer bone metastasis. In one embodiment, the assay comprisesapplying the composition or treatment to a SCID or other immunedeficient mouse receiving an intratibial injection of prostate cancerxenograft cells and determining the effect of the treatment on theprogression of bone metastasis by monitoring the tibial bone marrow forthe presence and levels of prostate cancer cells and/or osteoblasticmacrometastatic lesions. The presence of prostate cancer cells in bonemarrow may be detected by various means, including histologically,immunochemically, or by assaying for the presence of PSA mRNA orprotein. The presence of osteoblastic macrometastatic lesions may bedetected using histologic, radiographic, or other imaging techniques.This aspect of the invention may also have various clinicalapplications, including using the model in a method to assess theprognosis of a patient with locally advanced prostate cancer, and inparticular, to predict the likelihood that a patient will progress tometastatic disease. For example, in one embodiment, the method comprisesinjecting a single cell suspension prepared from a patient's prostatebiopsy material directly into the bone marrow of an immune deficientmouse and then monitoring the bone marrow for the development of bonelesions. The rate of bone lesion growth and osteoblastic activity may beused as prognostic indicators. The results of such analysis may assist atreating oncologist in determining how aggressively to treat a patientwith locally advanced disease.

Similarly, the effect of various therapeutic strategies for managinglocally advanced or metastatic disease in a particular patient may bepredicted. For example, the effect of a treatment strategy may bepredicted by applying the treatment to an immune deficient mousereceiving a bone marrow injection of the patient's prostate cancercells. The effect of the treatment may be monitored by comparing therate and extent of bone lesion growth and osteoblastic activity in thetest mouse to the corresponding rates in an untreated control mousereceiving a corresponding bone marrow injection. In addition, thismethod may be used to test the effectiveness of a treatment strategy onandrogen independent prostate cancer cells by using a female orcastrated male immune deficient mouse in order to select for androgenindependent clones in the patient's tumor material. The results of suchtests may assist a treating oncologist in determining which of severalalternative therapies should be used to manage a particular patient'sdisease.

Short-term Culture of Xenograft Tumor Cells

Xenograft tumor cells may be expanded using short-term in vitro tissueculture techniques well known in the art. In addition, different clonalpopulations from a xenograft tumor may be isolated through tissueculture techniques. In this regard, the invention provides methods forpreparing single cell suspensions from xenograft tumor tissue samples.In one embodiment, xenograft tumor tissue is surgically removed from asubcutaneous xenograft tumor, disaggregated, and proteolyticallydigested, using the method described in Example 2 or similar methods.Cells may then suspended in a solution of Matrigel, other basementmembrane compositions, saline, or other buffers. Such preparations areuseful for establishing new tumors in SCID or other immune deficientrecipient mice by, for example, subcutaneous inoculation, intraprostaticinjection, or by injection directly into bone marrow metaphyses. Cellsuspensions may be prepared from subcutaneous, intraprostatic, bone orother orthotopic tumors growing in SCID or other immune deficient mice.

Methods of Expanding and Purifying Prostate Cancer Cell Populations

Another aspect of the invention provides methods for expanding advancedstage prostate cancer cells, methods for preparing relatively purepopulations of prostate cancer cells from heterogeneous populations ofcells, and methods for propagating stage-specific prostate cancer cellsin vivo or in vitro. Primary tumor samples are heterogeneous in theircellular compositions, and are usually contaminated with normal andstromal cells. Moreover, it is difficult to obtain substantialpopulations of prostate cancer cells from human tissue biopsy material.In contrast, cells harvested from subcutaneous prostate tumors growingin SCID mice predominantly comprise prostate cancer cells. Thus, themodels of the invention provide a vehicle for purifying advanced stagehuman prostate cancer cells from heterogeneous biopsy material.

Serial passage of xenograft tumors in additional mice may be used tofurther enhance the prostate cancer specificity of the xenograftcellular composition. Similarly, the ability to serially propagate, suchas by serial propagation, relatively pure human prostate cancer cells inimmune deficient mice provides a means for obtaining large quantities ofdefined prostate cancer cells.

In one embodiment, tissue harvested from xenograft tumors is enrichedfor prostate cancer cells by subsequent passage in additional SCID orother immune deficient mice. In another embodiment, cells from xenografttumors may be cultured in vitro. In another embodiment, single cellsuspensions of prostate cancer cells may be prepared from such culturedcells or directly from xenograft tumor tissue. Single cell suspensionsprepared from protease digested subcutaneous xenograft tumors retain thebiological properties of the parental tumors. The single cellsuspensions may be used to establish, for example, new subcutaneoustumors, intraprostatic tumors, or bone tumors. As shown by theexperiments set forth in Example 2, as few as 10 xenograft cells canseed a new subcutaneous tumor.

Selective factors may be added to the environment in which the tumorcell enrichment is being conducted in order to expand cells with aparticular phenotype. For example, the presence of androgen in the invivo environment may be controlled by chemical or surgical castrationmethods well known in the art in order to select for androgen dependentor independent prostate cancer cells. Alternatively, female mice may beused to expand androgen independent cells. Similarly, in an in vitroenvironment, the absence of androgen in growth media may be used toselect androgen independent prostate cancer cells.

In addition, the presence of cell surface proteins on the tumor cells ofsubcutaneous xenografts may be used to distinguish and isolate humanprostate cancer cells from other cells. In particular, antibodies tocell surface proteins differentially expressed on prostate cancer cells(relative to their expression on murine marrow cells) may be used toisolate prostate cancer cells from xenograft tumor tissue, from cells inculture, etc., using antibody-based cell sorting or affinitypurification techniques. Most preferred for antibody-based cell sortingare antibodies to cell surface proteins which are human prostate cancerspecific. However, antibodies to other human proteins may be effectivelyemployed provided they do not exhibit significant cross-reactivity withthe murine homolog of the protein. An examples of such a protein ishuman galectin-6.

The ability to generate large quantities of relatively pure advancedstage human prostate cancer cells which can be grown in tissue cultureor as xenograft tumors in SCID or other immune deficient mice providesmany advantages, including, for example, permitting the evaluation ofvarious transgenes or candidate therapeutic compounds on the growth orother phenotypic characteristics of a relatively homogeneous populationof prostate cancer cells. Additionally, this feature of the inventionalso permits the isolation of highly enriched preparations of humanprostate cancer specific nucleic acids in quantities sufficient forvarious molecular manipulations. For example, large quantities of suchnucleic acid preparations will assist in the identification of raregenes with biological relevance to prostate cancer disease progression.

Another valuable application of this aspect of the invention is theability to analyze and experiment with relatively pure preparations ofviable prostate tumor cells cloned from individual patients with locallyadvanced or metastatic disease. In this way, for example, an individualpatient's prostate cancer cells may be expanded from a limited biopsysample and then tested for the presence of diagnostic and prognosticgenes, proteins, chromosomal aberrations, gene expression profiles, orother relevant genotypic and phenotypic characteristics, without thepotentially confounding variable of contaminating cells. In addition,such cells may be evaluated for neoplastic aggressiveness and metastaticpotential in the subcutaneous, orthotopic, and bone tumor models of theinvention. This aspect of the invention provides a means for testingalternative treatment modalities with a view towards customizingoptimal, patient-specific treatment regimens. Similarly,patient-specific prostate cancer vaccines and cellularimmunotherapeutics may be created from such cell preparations.

The prostate cancer models of the invention further provide methods forisolating stage-specific prostate cancer cells, includingmicrometastatic and osteoblastic prostate cancer cells. In oneembodiment, micrometastatic cells are isolated from hematopoetic tissuessuch as bone marrow or blood using antibody-based cell sorting oraffinity purification techniques. In another embodiment, osteoblasticprostate cancer cells are isolated from the bone marrow of SCID or otherimmune deficient mice bearing osteoblastic bone lesions. The presence ofsuch bone lesions may be detected histologically, immunohistochemically,or radiographically. Stage-specific prostate cancer cells may be furtherexpanded and purified by subsequent reimplantation into SCID or otherimmune deficient mice. For example, osteoblastic prostate cancer cellsmay be subpassaged in vivo by reinjection into bone marrow or in vitrousing defined bone stroma as a growth substrate.

As shown by the experimental work presented in Example 3, small numbersof micrometastatic prostate cancer cells can be detected in and isolatedfrom the bone marrow of SCID mice bearing subcutaneous prostate cancerxenografts. Although these prostate cancer cells represent less thanabout 1% of the cells in the bone marrow of the host mice, they may beisolated and expanded using cell purification methods, such as thosediscussed above. In one embodiment, bone marrow harvested from micebearing subcutaneous xenografts is incubated with a human specificmonoclonal antibody to galectin-6 and a secondary antibody conjugated tomagnetic beads. Prostate cancer cells are then isolated using MiltenyiMagnetic Minimacs columns (Sunnyvale, Calif.) to magnetically retainantibody-positive cells in the column while allowing antibody-negativecells to pass to the flow-through. Small numbers of micrometastaticprostate cancer cells isolated in this manner may be expanded in vivo bysubcutaneous inoculation of Matrigel suspended cells into SCID or otherimmune deficient mice. Osteoblastic prostate cancer cells may besimilarly isolated directly from bone marrow lesions.

The ability to purify and expand stage-specific prostate cancer cellsmay have various clinical applications. For example, stage-specificprostate cancer cells within clinical material may be isolated by usingcell sorting or purification techniques and then expanded assubcutaneous, intraprostatic: or bone tumors in SCID or other immunedeficient mice, depending on the particular objective. In oneembodiment, micrometastasis are isolated from patient serum, formulatedinto single cell suspensions, and injected into subcutaneously with theobjective of expanding these cells generally. In an alternativeembodiment, the micrometastatic cell preparation is injected into thebone marrow of a SCID or other immune deficient mouse with the objectiveof selectively expanding those cells with osteoblastic characteristics.Prostate cancer cells passaged in this manner may become conditioned byvarious factors within the microenvironment of the bone marrow and mayform osteoblastic lesions which may then be harvested for further use oranalysis.

Continuous Cell Lines

The invention also provides continuous human prostate cancer cell linesestablished by culturing xenograft cell preparations. In one embodiment,the cell line comprises human prostatic cancer cells cultured from asubcutaneous xenograft. In a specific embodiment described by way ofExample 9, the cell line LAPC-4 was established by culturing a singlecell suspension prepared from the LAPC-4 xenograft. The LAPC-4 cell lineexpresses PSA, androgen receptor (AR), and is androgen dependent. TheLAPC-4 cell line has been growing in continuous culture for 1.5 years,and retains phenotypic characteristics which correlate to the humanclinical situation more closely than any other available human prostatecancer cell line.

The cell lines of the invention may be used for a number of purposes. Byway of example and not by way of limitation, the cell lines may be usedas a source of large quantities nucleic acids and proteins, as a toolfor screening and evaluating candidate therapeutic transgenes, proteinsand compounds, and as a tool for identifying and isolating prostatespecific or differentially expressed genes. Genes which may regulate thegrowth of prostate cancer can be evaluated by overexpression intransduced cells grown in vitro or in vivo. The effects of genes onmicrometastasis and the development of osteoblastic bone lesions may beevaluated in vivo by subcutaneous, intraprostatic, or intratibialinoculation of transduced cells.

EXAMPLES

The invention is further described and illustrated by way of thefollowing examples and the experimental details therein. This section isset forth as an aid to understanding the invention, but is not intendedto, nor should it be construed as, limiting the invention as claimed.

Example 1

Generation of Subcutaneous Human Prostate Cancer Xenografts thatSimulate Prostate Cancer Progression

Materials and Methods

Patients: All clinical material was obtained from patients with locallyadvanced or metastatic (stage C or D) after obtaining informed consentaccording to an IRB approved protocol. Most patients had undergone someform of androgen ablation therapy (medical or surgical) and shownprogressive disease at the time the tissue samples were obtained.

Animals: C.B.17 scid/scid (SCID) mice were bred at UCLA under sterileconditions as previously described (Aldrovandi et al., Nature363:732-736 (1993)). Biopsy material obtained at the time of surgery wasplaced on ice and immediately transferred to the SCID mouse facility forimplantation. A scalpel was used to mince the tissue into 2-3 mm³sections which were then implanted subcutaneously into the flanks ofSCID mice. Mice were anesthetized with methoxyflurane beforeimplantation. Initial implants were performed with 100-200 μl ofMatrigel (Collaborative Research, Bedford, Mass.) injected around theimplant. Matrigel is an extracellular matrix preparation useful forenhancing the growth of epithelial tumors in vivo (Pretlow (1993),supra; Noel et al., Biochemical Pharmacology 43:1263-1267 (1992) and Limet al., Prostate 22:109-118 (1993)). Once a xenograft was passaged 2-3times, Matrigel was no longer used for serial propagation. Androgenablation was performed by surgical castration under anesthesia. Tumorsizes were determined by weekly caliper measurements of height, widthand depth. Sustained-release testosterone pellets (Innovative Researchof America, Sarasota, Fla.) were implanted subcutaneously, asrecommended by the manufacturer, in some experiments. Xenografts werestored viably in liquid nitrogen by freezing 1-2mm³ minced tissuesections in DMSO-containing medium.

PCR Assays, Histology and Immunochemistry: DNA from tumor tissue wasprepared using SDS detergent extraction and proteinase K digestion asdescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Edition 2 (1989). RNA was preparedby using a commercially available kit containing guanidine thiocyanateand β-mercaptoethanol (RNAgents Total RNA Isolation System, Promega). Toavoid contamination of gross tissue preparations, the tissue homogenizerand all surgical instruments used at the time of necropsy were cleanedby repeated rinses in HCl, DEPC treated water and ethanol. DNA-PCRassays for human β-globin (Aldrovandi et al., supra and Saiki et al.,Science 230:1350-1354 (1985)) and RT-PCR assays for PSA (Pang et al.,Hum. Gene Ther. 6:1417-1426 (1995)) were performed as previouslydescribed. Briefly, PCR analysis using primers specific for the humanβ-globin gene were performed for 30 cycles with 100 ng of genomic DNAisolated from the LAPC xenografts. One-tenth of each reaction mixturewas analyzed by electrophoresis through agarose gels and visualized bystaining for ethidium bromide. Murine 3T3 cells were used as a negativecontrol. The quality of all RNA samples was confirmed by ethidiumbromide staining for ribosomal RNA and by RT-PCR using primers forβ-actin (Pang et al., supra) as a control. Details on the primersequences can be found in the original references. RT-PCR analysis forPSA expression was performed on 100 ng of total RNA using primersspecific for human PSA. The same RNA samples were analyzed using primerswhich recognize human or murine β-actin to confirm equivalent loading ingels. Immunohistochemical staining for PSA was performed usingpolyclonal antisera to PSA (Dako) as described (Hsu et al., Am. J. Clin.Path. 75:734-738 (1981)).

Sequencing Androgen Receptor DNA: Exons 2-8 of the androgen receptorwere sequenced from genomic DNA using intron-specific PCR primers(Marcelli et al., Mol. Endocrinol. 90: 1105-1116 (1990)). PCR productswere initially screened by SSCP using appropriate positive and negativecontrols as described (Sutherland et al., J. Urol. 156: 828-831 (1996)).This technique has been shown to detect mutations in prostate cancerclinical specimens even when tumor cells represent only 20% of thepopulation used to make genomic DNA. All SSCP abnormalities wereanalyzed by sequencing. Two independent DNA samples were analyzed in twoindependent laboratories to rule out the presence of any mutations.

Cytogenetics: Tumor tissue was aseptically transported in DMEM growthmedium supplemented with 10% fetal bovine serum by overnight courier tothe University of Utah for cytogenetic preparation and analysis.Briefly, tissue was minced and washed in Hanks Balanced Salt Solution(Ca⁺⁺ and Mg⁺⁺ free), resuspended in RPMI medium supplemented with lotfetal bovine serum, and cells were arrested in metaphase with 0.001μg/ml colcemid for 16 hours. Cytogenetic harvests were carried out usingstandard procedures and, following hypotonic (0.075M) KC treatment and3:1 methanol/acetic acid fixation, slides were prepared and chromosomesG-banded with trypsin/Wrights stain.

Results

Advanced Stage Prostate Cancer Explants Can Be Serially Propagated inSCID Mice: Biopsies of locally advanced or metastatic tumor tissue wereobtained from a total of 15 patients with locally advanced or metastatic(stage C, D1 or D2) prostate cancer who underwent palliative surgicalprocedures due to complications from disease. Biopsy material wasimmediately transferred from the surgical suite to the SCID mousefacility, minced into 2-3 mm3 sections and implanted subcutaneously intoSCID mice in the presence of Matrigel. Tumor growth was scored positiveonly if the explant showed a sustained a two- to three-fold increase insize. In addition to histologic studies, two molecular assays wereperformed on each xenograft to verify the human origin of the tumors.These include a PCR assay on genomic DNA using primers specific for thehuman β-globin gene and a quantitative RT-PCR assay on RNA from tumorsusing primers specific for the human PSA gene. The PSA expression assaywas also used to verify the prostatic origin of the xenografts.

The results obtained from subcutaneous implantation of tumor tissuesamples from two separate series of these 15 patients are individuallydescribed below (i.e., the LAPC-1 through LAPC-8 series, and the LAPC-9through LAPC-1 5 series).

LAPC-1 through LAPC-8 Series:

Explants from six of eight patients (named LAPC 1-8 for Los AngelesProstate Cancer) formed tumors after a latent period which varied from2-10 months (Table 1). The six explants which grew were passaged intosecondary recipients in an attempt to establish permanent xenografts.Two of these (LAPC-1 and LAPC-5) were terminated after 3-4 passagesbecause we were unable to detect human DNA or expression of PSA in thetumors. These explants perhaps were overgrown by cells of murine originbecause they contained human DNA content and expressed PSA during earlypassages of LAPC-5 (Table 1, column 6,7).

The remaining four explants (LAPC-3,4,7 and 8) (LAPC-3 cell line,deposited with American Type Culture Collection, Manassas, Va., on March3, 2000, ATCC No. 1440) were successfully propagated as subcutaneousxenografts in secondary recipients for between 4 and 20 (or more)passages. RT-PCR was used to measure the levels of PSA mRNA expressionin comparison with LNCaP, a prostate cancer cell line known to expressPSA mRNA and protein. This assay is semi-quantitative and is capable ofdetecting PSA mRNA expression from 100 LNCaP cells diluted into 10⁵mouse cells (1 in 1000 or 0.1%) (FIG. 1B, top panel). Four of the sixxenografts (LAPC-3, 4, 5, 8) expressed human PSA at levels that variedfrom 1% to 100% of the level found in LNCaP cells (FIG. 1B, top panel).Simultaneous RT-PCR analysis using primers for β-actin confirmed thatequivalent levels of RNA were present in each reaction (FIG. 1B, bottompanel). FIG. 2 shows a histological comparison of the original LAPC-4tumor sample obtained at the time of surgery to the same tumor afterpassage as a xenograft in male mice. The hematoxylin and eosin stainedsections (FIG. 2, left panels) show a monotonous population ofanaplastic cells which stain positive for PSA using immunohistochemicalanalysis (FIG. 2, right panels). These findings demonstrate thatadvanced stage prostate cancer explants can be serially propagated inSCID mice and retain definitive tissue specific gene expression.

                                      TABLE 1                                     __________________________________________________________________________    SUMMARY OF IMPLANTS INTO SCID MICE/LAPC-1 THROUGH LAPC-8 SERIES.sup.1         Patient                                                                       Implant       Time interval                                                   (disease                                                                           Biopsy   for tumor   Human DNA                                           stage)                                                                             site Growth                                                                            growth Passages                                                                           Status.sup.2                                                                         PSA Status.sup.3                                                                     Notes                                 __________________________________________________________________________    LAPC-1                                                                             liver met                                                                          yes 2 months                                                                             5    negative on                                                                          negative                                                                             overgrowth by                         (stage D)                 passage       tumor of                                                                      murine origin                                                                 after serial                                                                  passage                               LAPC-2                                                                             lymph                                                                              no  no growth at 2                                                                       1    --     --     --                                    (stage D)                                                                          node     years                                                                met                                                                      LAPC-3                                                                             prostate                                                                           yes 10 months                                                                            3    positive                                                                             positive                                                                             no PSA                                (stage D)                                                                     channel                          positive cells                                    TURP                               outside site of                                                               implantation                                                                  (n = 2)                               LAPC4                                                                              lymph                                                                              yes 3 months                                                                             >8   positive                                                                             positive                                                                             PSA positive                          (stage D)                                                                          node                               cells in bone                              met                                marrow,                                                                       spleen, blood                                                                 in 50% of mice                                                                (n = 12)                              LAPC-5                                                                             lymph                                                                              yes 9 months                                                                             5    positive, then                                                                       positive, then                                                                       overgrowth by                         (stage D)                                                                          node                 negative on                                                                          negative on                                                                          tumor of                                   met                  passage 4                                                                            passage 4                                                                            murine origin                                                                 on passage 4                          LAPC-6                                                                             prostate                                                                           no  no growth at 9                                                                       1    --     --     --                                    (stage C)     months                                                          LAPC-7                                                                             prostate                                                                           yes 3 months                                                                             2    positive                                                                             negative                                                                             --                                    (stage C)                                                                     LAPC-8                                                                             lymph                                                                              yes 10 months                                                                            2    positive                                                                             positive                                                                             no PSA                                (stage D)                                                                          node                               positive cells                             met                                outside                                                                       implantation                                                                  site (n = 1)                          __________________________________________________________________________     .sup.1 locally advanced (stage C) or metastatic (stage D) disease;            .sup.2 determined by PCR of genomic DNA for human globin;                     .sup.3 determined by RTPCR and/or immunohistochemistry                   

Two of the xenografts in this series, LAPC-3 and LAPC-4, have retainedconstant histologic and molecular features of prostate cancer for morethan 6 and 8 passages, respectively. Both xenografts can be frozenviably as tumor explants and recovered from freezing with nearly 100%efficiency. A cell line from the LAPC-4 xenograft was established byserial passage of trypsinized, minced xenograft tissue in Iscove'sgrowth medium supplemented with 20% fetal calf serum. The LAPC-4 cellline has remained established for more than 20 passages and has been incontinuous culture for over 18 months. These cells continue to expressPSA, form tumors in SCID mice, and retain androgen-responsiveness.

LAPC-9 through LAPC-15 Series:

A second series of xenograft experiments was conducted by implantingtissue samples from an additional seven prostate cancer patients withadvanced stage (C or D) disease (Table 2). Four of these seven implantshave resulted in the generation of androgen-responsive xenografts whichexpress PSA and which are capable of being propagated serially inadditional mice (LAPC-9, 12,14,15). The LAPC-9 xenograft (LAPC-9 cellline deposited with American Type Collection, Manassas, Va., on Mar. 3,2000ATCC No. 1442), generated from a bone tumor biopsy of a patient withhormone-refractory metastatic disease, demonstrates an extremelyandrogen-sensitive phenotype (PSA levels drop to zero after castration)and has been passaged and viably maintained for about 1 year. TheLAPC-14 xenograft, generated from a prostate tumor biopsy of a patientwith metastatic disease, demonstrates aggressive growth characteristicsand exhibits a high degree of androgen-responsiveness (growth issubstantially enhanced by the addition of testosterone).

                                      TABLE 2                                     __________________________________________________________________________    SUMMARY OF IMPLANTS INTO SCID MICE/LAPC-9 THROUGH LAPC-15 SERIES.sup.1        Patient         Time                                                          Implant         interval for                                                                             Human                                              (disease        tumor      DNA PSA                                            stage)                                                                              Biopsy site                                                                         Growth                                                                            growth                                                                              Passages                                                                           Status.sup.2                                                                      Status.sup.3                                                                      Notes                                      __________________________________________________________________________    LAPC-9                                                                              femur yes  5 weeks                                                                            5    positive                                                                          positive                                                                          patient hormone-                           (stage D)                                                                           tumor                        refractory                                                                    androgen                                                                      dependent                                  LAPC-10                                                                             prostate                                                                            no  --    --   --  --  Gleason 9                                  (stage C/D)                                                                         (prostat-                                                                     ecomy)                                                                  LAPC-11                                                                             femur no  --    --   --  --  1 week post lupron                         (stage D)                                                                           tumor                        2 weeks post                                                                  flutamide                                  LAPC-12                                                                             prostate                                                                            yes 13 weeks                                                                            1    positive                                                                          positive                                                                          metastatic                                 (stage D)                                                                           TURP                         hormone                                                                       refractory                                 LAPC-13                                                                             trans-                                                                              no  --    --   --  --  Gleason 7                                  (stage C/D)                                                                         rectal                                                                        biopsy                                                                  LAPC-14                                                                             prostate                                                                            yes  4 weeks                                                                            2    positive                                                                          positive                                                                          metastatic                                 (stage D)                                                                           TURP path                    PSA < 0.2                                        neg                          lupron treated                                                                androgen                                                                      responsive                                 LAPC-15                                                                             prostate                                                                            yes 12 weeks                                                                            1    positive                                                                          positive                                                                          metastatic to bone                         (stage D)                                                                           TURP                         lupron treated                                                                casodex treated ×                                                       4 mos                                      __________________________________________________________________________     .sup.1 locally advanced (stage C) or metastatic (stage D) disease;            .sup.2 determined by PCR of genomic DNA for human globin;                     .sup.3 determined by RTPCR and/or immunohistochemistry                   

LAPC-3 and LAPC-4 Xenografts Contain Chromosome Abnormalities:

Extensive cytogenetic studies of human prostate cancer have beendifficult due to heterogeneity of clinical material obtained at surgeryand limited growth of prostate tumor cells in vitro. To determine ifpassage of prostate tumor tissue in SCID mice might facilitatekaryotypic analysis, early passage tumors from the LAPC-3 and LAPC-4xenografts were analyzed using standard cytogenetic techniques. A highmitotic yield was obtained from tumor samples from both xenografts andall metaphase cells contained human chromosomes. Detailed compositekaryotypes are noted in Table 3. The modal chromosome number of LAPC-4was 89, suggesting a hypotetraploid line, whereas the modal chromosomenumber of LAPC-3 was 69, which suggests that this line is near-triploid,yet the presence of four copies of many chromosomes raises thepossibility of reduction from tetraploid. Both xenografts showpreviously reported numerical and structural chromosome abnormalitiessuch as loss of Y and 16. In addition, both xenografts contain adeletion at chromosome 12p12, a karyotypic abnormality that has not beenpreviously reported in prostate cancer.

                                      TABLE 3                                     __________________________________________________________________________    CYTOGENETIC ANALYSIS OF LAPC-3 AND LAPC-4 XENOGRAFTS                               Passage number at                                                             time of analysis                                                                       Number of                                                                           Modal                                                          (number of                                                                             metaphases                                                                          Chromosome                                                Xenograft                                                                          independent tumors)                                                                    analyzed                                                                            Number                                                                              Karyotype                                           __________________________________________________________________________    LAPC-3                                                                             passage 2 (1 tumor);                                                                   80    69    68-81, XXY + add(I)(p22), -2, +3, +4,                    passage 3 (2 tumors) +5, del(6)(q21) × 2, +7, +9, +9, -11,                                   del(12)(p12), -13, -13, +14,                                                  t(14;14)(q10;q10), -16, +18, +19, +20                                         [cp80]                                              LAPC-4                                                                             passage 3 (2 tumors)                                                                   44    89    76-92, XX, -Y, -Y, add(8)(p23), +9,                                           del(12)(p12), -14, -16, -18, -21, +mar1,                                      +mar2[cp44]                                         __________________________________________________________________________

Progression of LAPC-4 Xenograft to Androgen Independence:

Prostate cancer cells are exquisitely sensitive to the growthstimulatory effects of androgen, but androgen-independent diseaseeventually develops in patients under the selective pressure of androgendeprivation. The mechanism for this transition to androgen-independentgrowth is unknown. The question of whether this phase of the diseasecould be modeled in SCID mice was determined using the LAPC-4 xenograft,which reproducibly forms tumors on serial passages in male mice with100% frequency. The androgen dependence of the xenograft was measured invivo by comparing the growth rates after implantation in intact malemice with those from castrated male mice or female mice. For LAPC-4, theaverage time for tumor formation in castrated male mice or female mice(n=10) was 13.4 weeks versus 4.3 weeks in intact males (n=14) (FIG. 3).The delayed growth in female mice was reversed by implantation of a90-day sustained-release testosterone pellet (FIG. 3). Theandrogen-independence of tumors growing in female or castrated male micewas confirmed by secondary transfer experiments. Once established, thesetumors grew within 4-5 weeks in male, female and castrated male mice.

The LAPC-3 xenograft showed growth characteristics similar to theandrogen independent sublines of LAPC-4. After an initial latent periodfor passage 1, LAPC-3 tumors grew within 7-8 weeks regardless of thehormonal background of the recipient (FIG. 3), clearly establishing thisxenograft as androgen independent.

Clinically, anti-androgen therapy causes temporary regression of diseasein most patients with advanced prostate cancer. To determine if asimilar phenomenon is observed in the mouse model, the effect of acuteandrogen deprivation on established tumors growing in male mice wasexamined. Equivalent size implants of the LAPC-4 xenograft were passagedinto a cohort of 14 male mice, all of which developed easily measurabletumors after four weeks. Half of these mice underwent castration, thenthe tumor sizes in each group were determined weekly by calipermeasurement of tumor diameters in three dimensions. Tumors in theuncastrated mice doubled in size over a 2-3 week period (FIG. 4). Incontrast, the castrated mice showed a decrease in tumor size at one weekof approximately 50 percent which persisted for 2-3 weeks. These tumorsresumed growth after a variable latent period (3-8 weeks) and eventuallygrew to the same size seen in uncastrated mice. These results show thatthe LAPC-4 xenograft displays androgen-dependent growth, thatandrogen-independent sublines can be developed, and that this xenograftsimulates the clinical transition from androgen-sensitive toandrogen-independent disease.

LAPC-3 and LAPC-4 Express Wild-Type Androgen Receptors:

To determine whether similar mutations are present in LAPC-3 and LAPC-4,exons 2-8 of the androgen receptor gene, which span the DNA binding andligand binding domains of the receptor, were sequenced. Single-strandconformational polymorphism (SSCP) analysis was also performed. Eachexon was amplified by PCR from genomic DNA of early and late passagetumors and analyzed using previously characterized mutant and wild-typeandrogen receptor DNA as positive controls (Sutherland et al., 1996).The results show that both LAPC-3 and LAPC-4 contain wild-type sequencesin exons 2-8. Furthermore, these sequences remain wild-type in androgenindependent LAPC-4 sublines. Immunoblot analysis confirmed expression ofandrogen receptor protein of the appropriate size. These results providedefinitive evidence that androgen independent prostate cancerprogression can occur in the absence of androgen receptor mutations inthe DNA or ligand binding domains.

LAPC-4 Cells Can Be Efficiently Transduced With Retroviruses: LAPC-4xenograft cells can be successfully transduced by retroviruses packagedtransiently in 293T cells with an amphotropic envelope protein. LAPC-4cells were infected with retrovirus stocks expressing the cell surfaceThy-1 protein and expression detected by flow cytometry using anantibody to Thy-1. The results showed Thy-1 expression in up to 50% ofthe cells 48 hours after infection, indicating successfulretroviral-mediated transduction of the Thy-1 gene into LAPC-4 cells.

Example 2

Preparation of Single Cell Suspensions of Xenograft Cells

Materials and Methods

Single cell suspensions of subcutaneous LAPC-4 tumors were prepared asfollows. After removing xenograft tissue from SCID mouse, tissue wasminced into 1-2 mm³ sections while the tissue was bathed in 1× Iscovesmedium, minced tissue was then centrifuged at 1.3K rpm for 4 minutes,the supernatant was resuspended in 10 ml ice cold 1× Iscoves medium andcentrifuged at 1.3K rpm for 4 minutes. The pellet was then resuspendedin 1× Iscoves with 0.1% pronase E and incubated for 18 minutes at roomtemperature with mild rocking agitation followed by incubation on icefor 2-4 minutes. The mixture was then filtered using a 200 μm nylon meshfiler. Filtrate was centrifuged at 1.3K rmp for 4 minutes, and thepronase was removed from the aspirated pellet by resuspending in 10 mlIscoves and re-centrifuging. Resulting pellets were resuspended in PrEGMpre-incubated at 37 degrees C. Cell counts were determined, and limitingdilutions were formulated as indicated in FIG. 5.

Results

The results of a limiting dilution analysis of tumor engraftment usingsingle cell suspension of LAPC-4 xenograft cells are shown in (FIG. 5).The results show that single cell suspensions of xenograft cells canform subcutaneous tumors in male mice after injection of as few as 10LAPC-4 cells and that these cells retain the androgen responsiveness ofthe parental tumors.

Example 3

Simulation of Progression to Micrometastasis in SCID Mice BearingSubcutaneous Tumors

Materials and Methods

The LAPC-4 xenograft was used in this study. This xenograft was derivedfrom a lymph node containing metastatic prostate cancer cells, and 100%of male mice inoculated subcutaneously with LAPC-4 cells developlocalized tumors after 4-6 weeks without evidence of bony metastasis.The presence of micrometastasis in SCID mice implanted with LAPC-4tumors was determined by analyzing the peripheral blood for prostatecancer cells using RT-PCR assays for PSA mRNA. Simultaneous RNA-PCRstudies using β-actin primers demonstrated equivalent RNA loading. Toconfirm that positive PSA mRNA signals were not due to contaminationwith tumor cells during the necropsy procedure of during the preparationof RNA, samples were simultaneously prepared from a control mouse thatwas not implanted with a xenograft. No PSA expression was detected incontrol mice, even after prolonged autoradiograph exposure times (FIG.6). Bone marrow, spleen, liver, lung and kidney tissue from miceimplanted with subcutaneous LAPC-4 tumors was also analyzed for thepresence of prostate cancer cells using RT-PCR to detect PSA mRNA.

Results

Examples of the analysis from two mice (FIG. 6A, mouse nos. 213 and 241)demonstrate detection of PSA mRNA in blood at a 0.1-1.0% level, which iscomparable to levels reported in clinical studies. Other organs werepositive in several mice, including bone marrow (mouse 213, 241), lung(mouse 214), and spleen (data not shown). The results from 12 animalsbearing LAPC-4 xenografts (Table 4) show that 50 percent of mice havePSA mRNA positive cells (level of PSA expression by RT-PCR of 0.1percent or greater) detected in peripheral blood, bone marrow or spleen.The level of expression was roughly quantitated by comparison to aseries of LNCaP cells diluted into murine fibroblasts and varied from0.1% to 1.0%. It is of interest that the frequency of detectingmicrometastatic disease was higher (80%) in female mice or in male micecastrated prior to implantation compared to intact males (27%). Theseresults suggest that the transition to androgen-independent disease isassociated with a higher metastatic rate, a hypothesis which is alsosupported by clinical experience.

                  TABLE 4                                                         ______________________________________                                        FREQUENCY OF DETECTION OF PSA POSITIVE CELLS IN                               HEMATOPOETIC TISSUES OF LAPC-4 BEARING SCID MICE                                       Number of mice with PSA positive cells in hematopoietic              Group    organs per total number analyzed                                     ______________________________________                                        Intact Males                                                                           2/7 (29%)                                                            Castrate Males                                                                         4/5 (80%)                                                            (or Females)                                                                  Total    6/12 (50%)                                                           ______________________________________                                    

Example 4

Generation of Intraprostatic Tumors with Xenograft Cells

Materials and Methods

Single cell suspensions were prepared from subcutaneous xenografts asdescribed in Example 2. SCID mice were anesthetized withKetamine/Xylazine prior to implantation. Transverse incisions were madein the lower abdomen of mice, abdominal wall muscles were incised, andthe bladder and seminal vesicles were delivered through the incision toexpose the dorsal prostate. Approximately 10,000 LAPC-4 suspended in 10μl PrEGM were slowly injected into the dorsal prostate under the capsulevia a 30 gauge needle, and the incisions closed using a running suture.

Results

Intraprostatic injection of single cell suspensions prepared from theLAPC-4 and LAPC-9 xenografts and from the LAPC-4 cell line resulted inorthotopic tumors in recipient SCID mice with 100% efficiency.

Example 5

Simulation of Progression to Metastatic Stage of Prostate Cancer in SCIDMice Bearing Intraprostatic Tumors

Materials and Methods

Single cell suspensions of LAPC-4 xenograft cells were prepared and usedto establish orthotopic tumors in the prostates of SCID mice asdescribed in the preceding example. The presence of metastases weredetermined by histologic examination and by RT-PCR to detect PSA mRNAbetween 8 and 12 weeks post-injection.

Results

The results, shown in Table 5 below, indicate high frequencies of lymphand pulmonary metastasis as well as a significant frequency of bonemarrow metastasis formation. An enhanced frequency of bone metastasiswas observed in a subset of the mice pretreated with a combination ofradiation and NK cell depletion. Similar results were obtained using theLAPC-9 xenograft.

                  TABLE 5                                                         ______________________________________                                        PATTERN OF METASTASIS AFTER ORTHOTOPIC                                        INJECTION OF LAPC-4                                                           TUMOR SITE       FREQUENCY                                                    ______________________________________                                        Local Tumor      100%                                                         Pelvic Lymph Nodes                                                                             90%                                                          Lung             90%                                                          Bone Marrow      30%                                                          ______________________________________                                    

Example 6

Simulation of progression to Osteoblastic Bone Metastasis in SCID MiceInoculated Intratibially with Single Cell Suspensions of Xenograft Cells

Materials and Methods

Tibial Injection Assay: Prostate cancer cells were isolated from asubcutaneous xenograft LAPC-4 tumor and prepared as a single cellsuspensions as described in Example 2. Ten thousand LAPC-4 cellssuspended in 1 μl Matrigel were surgically injected into each proximaltibial metaphyses of a cohort of SCID mice via a 27 gauge needle. Threemice were sacrificed at each of 2, 4, 6, 8 and 12 weeks post injection.Serum PSA levels were periodically assayed by ELISA. At 2 weeks, frozenbone sections were analyzed immunohistochemically for cytokeratin-18staining with an antibody specific for human cytokeratin-18 or anisotype control antibody. Longitudinal sections of tibias from micesacrificed at 4, 6 and 8 weeks were analyzed for tumor growth byhematoxylin and eosin (H+E) staining of decalcified paraffin sections.Radiographs of mice were taken at necropsy to monitor evidence ofosteoblastic bone lesions.

Results

At 2 weeks, small numbers of human prostate cancer cells were visualizedby immunohistochemical staining with anti-cytokeratin-18 antibody (FIG.7). Cytokeratin-18 positive cells were observed scattered throughout themedulary cavity. This data indicates that the majority of LAPC-4 cellsinjected into the mouse tibia either die or migrate to other locationssince only a small subset of the injected cells can be detected at thistime point.

At 4 weeks, small foci of tumor growth were observed in a few isolatedareas, usually adjacent to normal bone spicules, by H+E histology (FIG.8A) and PSA could be detected in serum. At the 6 and 8 week time points,more extensive tumor growth throughout the marrow cavity was observedtogether with a progressive increase in new bone formation indicative ofosteoblastic activity within the marrow cavity in response tosurrounding tumor cells (FIGS. 8B and C). Serum PSA levels were markedlyelevated at this time point.

By 8 weeks, bone lesions were visible radiographically as a mixture ofosteoblastic and osteolytic lesions with dominant bone formation similarto clinical observations in human prostate cancer. Referring to FIG. 9,the left panel shows a radiograph of a normal mouse tibia with sharp,well defined cortex and relatively radioopaque marrow cavity. The rightpanel is a radiograph of the marrow cavity of the tibia injected withLAPC-4 xenograft cells, showing a heterogeneous increase in bone densitydue to osteoblastic activity and destruction of one area of the cortex.These results indicate that LAPC-4 xenograft cells can proliferate inmurine bones, suggesting that the crosstalk between bone stroma andprostate cancer cells can occur across species.

Example 7

Isolation of Prostate Cancer Cells from Bone Marrow of SCID Mice BearingSubcutaneous Xenografts

The presence the cell surface protein galectin-6 on LAPC-4 cells wasestablished by incubating intact LAPC-4 cells with a human specificmonoclonal antibody to galectin 6 or isotype control. The antibody wasvisualized by flow cytometry following incubation with a secondaryantibody conjugated to FITC. The flow cytometry results show expressionof galectin-6 at a level that is at least one order of magnitude abovebackground (FIG. 10). Similar experiments performed on mouse bone marrowshowed no galectin staining.

As described in Example 3, small numbers of prostate cancer cells can bedetected in the bone marrow of SCID mice bearing subcutaneous xenograftsat 4-6 weeks post-inoculation, representing somewhat less than 1% of thecells in the marrow. This population of prostate cancer cells may beisolated from bone marrow using Miltenyi Magnetic Minimacs (Sunnyvale,Calif.) antibody-based affinity purification system and anti-galectin-6antibody as follows. Twenty mice bearing subcutaneous LAPC-4 tumors areeuthanized 4-6 weeks after implantation of xenografts. Bone marrow isharvested from the tibias and femurs by flushing marrow cavities withsaline. Marrow is pooled and incubated with a human specific monoclonalantibody to galectin-6 and a secondary antibody conjugated to magneticbeads and run through the Minimacs column as recommended by themanufacturer. LAPC-4 cells will be retained on the column, while mousebone marrow cells will pass through. Purified LAPC-4 cells may then beharvested from the column and expanded by seeding subcutaneous tumors inSCID mice.

Example 8

Isolation of Prostate Cancer Cells from Bone Marrow of SCID MiceInjected Intratibially with Xenograft Cells

Intratibial tumors are established in SCID mice using LAPC-4 cells asdescribed in Example 6. LAPC-4 cells growing in bone marrow arerecovered from mice after necropsy at 12 weeks by flushing the tibialmarrow cavity with saline and harvesting the cells. At 12 weekspost-injection, about 90% of the recovered cells are prostate tumorcells with some residual murine bone marrow cells. This population ofcells may be further purified for prostate cancer cells using agalectin-6 antibody/magnetic affinity purification approach as describedin Example 7.

Example 9

LAPC-4 Cell Line Retains Expression of PSA, Androgen Receptor, andProstatic Acid Phosphatase Through Multiple Passages

Materials and Methods

A continuous cell line was established from the LAPC-4 xenograft byserial passage of trypsinized, minced xenograft tissue in Iscove'sgrowth medium supplemented with 20% fetal calf serum.

Results

LAPC-4 cells growing in continuous culture in vitro have retainedexpression of PSA, androgen receptor, and prostatic acid phosphatasethrough more than 20 passages. In addition, LAPC-4 cells contain nomutations in either the DNA or ligand binding domains of the androgenreceptor, which is a novel characteristic among known prostate cancermodels. The only other PSA-expressing cell line, LNCaP, expresses anandrogen receptor with a point mutation in the ligand binding domain. Inaddition, LAPC-4 cells continue to express androgen receptor in androgenindependent sublines, analogous to results obtained from the analysis ofclinical material. The LAPC-4 cell line is androgen dependent sincetumors grow rapidly in male mice but not in female or castrated malemice. The LAPC-4 cell line has remained established for more than 20passages and has been in continuous culture for over 18 months. Thesecells continue to express PSA, form tumors in SCID mice, and retainandrogen-responsiveness.

Example 10

Testing the Biological Effects of Candidate Genes on AndrogenIndependent Growth in vivo

Some genes upregulated in hormone refractory prostate cancer maycontribute to the pathogenesis of androgen independence. Bcl-2, forexample, which is upregulated in many advanced prostate cancers, hasbeen demonstrated to confer androgen independence to androgen dependentLNCaP prostate cancer cell line (Raffo et al., 1995). In accordance withthis example, one can access in vivo the contribution of candidate genesto the androgen independent phenotype.

LAPC-4 Androgen Dependent Tumor Explants Grow in Tissue Culture and FormAndrogen Dependent Tumors Upon Reinjection into SCID Mice

Current bioassays for androgen dependent and independent growth relyalmost exclusively on the LNCaP prostate cancer cell line, because it isthe only cell line available which displays features of androgendependence. In order to circumvent the problem of long-term passagedcell line with the potential for multiple in vitro mutations, the LAPC-4xenograft was grown in short term culture and then reinjected into miceto form tumors. Explanted tumors were then manipulated genetically andthe effects of these manipulations were measured in vivo.

LAPC-4 tumors were minced into small pieces and cultured in media with15% fetal calf serum. Outgrowth of both epithelial cells and fibroblastswas noted after 2-3 days. Cells then grew to confluence and could besuccessfully passaged to remove the original tumor pieces. RT-PCRconfirmed continued PSA expression. 1×10⁷ cells were then reinjectedinto either intact male or castrated SCID mice. Similar to the initialexperiments, injected cells formed tumors in an androgen dependentfashion, requiring prolonged periods to form tumors in castrated mice.

LAPC-4 Cultures can be Transduced with Retrovirus

In order to test the infectability of explanted LAPC-4 cells byretrovirus, these cells were transduced with a retroviral vectorcontaining a truncated nerve growth factor receptor gene (NGFR). A PG13packaging cell line, containing the gibbon-ape leukemia virus (GALV)envelope, was used to generate high titer virus. Retrovirus virionsproduced in this manner have the unique property of infecting human, butnot murine, cells, thus avoiding introduction of transgene into mousestromal cells (Bauer et al., 1995). After infection, the cells werestained with an antibody directed against NGFR and analyzed by FACSanalysis. Five-10% of cells were transduced. Murine fibroblasts negativecontrols showed no infection, while human 293T cells were efficientlytransduced.

Biological Assays for cDNAs Upregulated in Androgen-independent ProstateCancer

Candidate cDNAs can be cloned into the 5' position of the retroviralvector pSRalpha used extensively in our laboratory (Afar et al., 1994).A reporter gene, either NGFR, LacZ, or human codon-optimized greenfluorescent protein (GFP), would be inserted downstream. The plasmid canbe transfected into the PG13 packaging cell line, virus collected, andtiters measured. LAPC-4 cells can be infected after the first passageand then expanded without selection until sufficient numbers areavailable for injection. Transgene expression can be confirmed either byFACS analysis or by northern blot analysis using the RDA cDNA clone as aprobe.

Two different types of experiments can be performed. In the first,infected cells are injected in to the flanks of intact male SCID mice.After tumors form in both flanks of an individual mouse, one tumor isremoved and the mouse is then castrated. The explanted tumor is analyzedto quantify the percentage of cells infected. This can be done either byLacZ straining or by FACS analysis for GFP or NGFR. We anticipate that5-10% of cells will carry the transgene. The remaining tumor can besimilarly analyzed after it regresses and regrows (i.e., about 4-8 weeksafter castration). If the transgene confers a survival advantage orandrogen independence to infected cells, we would expect to see thepercentage of cells carrying the transgene to increase after hormoneablation. Multiple mice can be injected with each construct and positiveresults confirmed by repetition.

In a second set of experiments, one can implant infected cells intointact and castrated male mice in parallel after quantifying infectionfrequency. Resulting tumors (at 4 and 12 weeks, respectively) areanalyzed for insert frequency as described above. Again, we expect that"androgen independent" genes will provide an androgen independent growthadvantage and predominate in the resulting tumor. In addition, it ispossible that a given candidate gene will shorten the time to tumorformation in castrated males. This can also be measured. Finally, it ispossible that a given gene could cause aggressive androgen dependentgrowth. This too can be quantified in this assay, by comparing time totumor formation and insert frequency before and after injection intointact male mice.

These assays can be validated with positive controls. In particular, onecan use bcl-2, c-myc, and c-met, since these have been consistentlyassociated with androgen independence.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

References

Aldrovani, G. M. et al. The SCID-hu mouse as a model for HIV-1infection. Nature 363: 732-736 (1993).

Baston, O. V. The role of the vertebral veins in metastatic processes.Ann Int. Med. 16: 38-45 (1942).

Berettoni, B. A. and Carter, J. R. Mechanisms of cancer metastasis tobone. J. Bone Joint Surg. 68A: 308-312 (1986).

Brandt, B. et al. Isolation of prostate-derived single cells and cellclusters from human peripheral blood. Cancer Res. 56: 4556-4561 (1996).

Coman, D. R. and DeLong, R. P. The role of the vertebral venous systemin the metastasis of cancer to the spinal column. Cancer 4: 610-618(1951).

Deguchi, T. et al. Detection of micrometastatic prostate cancer cells inthe bone marrow of patients with prostate cancer. Brit. J. Cancer 75:634-638 (1997).

Fidler, I. J. Critical factors in the biology of human cancermetastasis. Cancer Res. 50: 6130-6138 (1990).

Ghossein, R. A. et al. Detection of circulating tumor cells in patientswith localized and metastatic prostate carcinoma: Clinical implications.J. Clin. Oncol. 13: 1195-2000 (1995).

Gleave, M. E. et al. Serum prostate specific antigen levels in micebearing human prostate LNCaP tumors are determined tumor volume andendocrine and growth factors. Cancer Res. 52: 1598-1605 (1992).

Haq, M et al. Rat prostate adenocarcinoma cells disseminate to bone andadhere preferentially to bone marrow-derived endothelial cells. CancerRes. 52: 4613-4619 (1992).

Hsu, S. M. et al. A comparative study of the peroxidase-antiperoxidasemethod and an avidin-biotin complex method for studying polypeptidehormones with radioimmunoassay antibodies. Am J. Clin. Pathol. 75:734-738 (1981).

Kaighn, M. E. et al. Establishment and characterization of a prostaticcarcinoma cell line. Invest. Urol. 17: 16-23(1979).

Karp, J. E., et al. Prostate cancer Prevention: Investigationalapproaches and opportunities. Cancer Res. 56: 5547-5556 (1996).

Katz, A. E. et al. Molecular staging of prostate cancer with the use ofan enhanced reverse transcriptase-PCR assay. Urology 43: 765-775 (1994).

Katz, A. E. et al. The role of reverse-transcriptase polymerase chainreaction assay for prostate-specific antigen in the selection ofpatients for radical prostatectomy. Urology Clinics of North America 23:541-549 (1996).

Kozlowski, J. M. et al. Prostate cancer and the invasive phenotype:Application of new in vivo and in vitro approaches. In: TumorProgression and Metastasis, edited by Fidler, I. J. and Nicholson, G.Alan R. Liss, Inc., New York, 1988, p. 189-231.

Lee, C. et al. In vitro and in vivo approaches to study metastasis inhuman prostatic cancer. Canc. Met. Rev. 12: 21-28 (1993).

Lim, D. J. et al. Growth of an androgen-sensitive human prostate cancercell line, LNCaP, in nude mice. Prostate 22: 109-118 (1993).

Liu, A. Y. et al. Prostatic cell lineage markers: Emergence of BCL2+cells of human prostate cancer xenograft LuCaP 23 following castration.Intl. J. Cancer 65: 85-89 (1996).

Maricelli, M. et al. Definition of the human androgen receptor genestructure permits the identification of mutations that cause androgenresistance: Premature termination of the receptor protein at amino acidresidue 588 cause complete androgen resistance. Mol. Endocrinol. 909:1105-1116 (1990).

Melchior, S. W. et al. Clinical relevance of prostate cells in the bonemarrow of patients with clinically localized carcinoma of the prostate(CAP). J. Urology 157: 1718 (1997 (Abstract).

Netland, P. A. and Zetter, B. R. Metastatic potential of B16 melanomacells after in vitro selection for organ-specific adherence. J. CellBiol. 10: 720-724 (1985).

Nishijima, Y. et al. Clinical significance of the vertebral vein inprostate cancer metastasis. Adv. Exp. Med. Biol. 324: 93-100 (1992).

Noel, A. et al. Basement membrance components (Matrigel) promote thetumorigenicity of human breast adenocarcinoma MCF7 cells and provide anin vivo model to assess the responsiveness of cells to estrogen.Biochemical Pharmacology 43: 1263-1267 (1992).

Olsson, C. A. et al. Reverse transcriptase-polymerase chain reactionassays for prostate cancer. Urology Clinics of North America 24: 367-378(1997).

Pang, S. et al. Prostate tissue specificity of the prostate-specificantigen romoter isolated from a patient with prostate cancer. Hum. GeneTher. 6: 1417-1426 (1995).

Pretlow, T. G. et al. Transplantation of human prostatic adenocarcinomainto nude mice in Matrigel. Cancer Res. 51: 3814-3817 (1991).

Saiki, R. K., et al. Enzymatic amplification of β-globin genomicsequences and restriction site analysis for diagnosis of sickle cellanemia. Science 230: 1350-1354 (1895).

Sambrook, J. et al. Molecular Cloning: A Laboratory Manual, Ed.2. ColdSpring Harbor Press, Clod Spring Harbor, New York, 1989.

Seiden, M. V. et al. Detection of circulating tumor cells in men withlocalized prostate cancer. J. Clin. Oncol. 12: 2364-2639 (1994).

Shevrin, D. H. et al. Development of skeletal metastasis by humanprostate cancer in athymic nude mice. Clin. Exp. Metastasis 6: 401-409(1988).

Shevrin, D. H. et al. Patterns of metastasis by the human prostatecancer cell line PC-3 in athymic nude mice. The Prostate 15: 187-194(1989).

Sutherland. R. W. et al. androgen receptor gene mutations are rarelyassociated with isolated penile hypospadias. J. Urol. 156: 828-831(1996).

Thalmann, G. N. et al. Androgen-independent cancer progression and bonemetastasis in the LNCaP model of human prostate cancer. Cancer Res. 54:2577-2581 (1994).

Wang, M. and Stearns, M. E. Isolation and characterization of 3 PC-3prostatic tumor sublines which preferentially metastasize to selectorgans in SCID mice. Differentiation 48: 115-125 (1991).

Wainstein, M. A. et al. Androgen-Dependent xenograft model derived froma primary human prostatic carcinoma. Cancer Res. 54: 6049-6052 (1994).

Wood, D. P. et al. Identification of bone marrow micrometastases inpatients with prostate cancer. Cancer 74: 2533-2540 (1994).

Zetter, B. R. et al. The cellular basis for prostate cancer metastasis.Adv. Exp. Med. Biol. 324: 39-43 (1992).

What is claimed is:
 1. A method of simulating the progression of humanprostate cancer from primary tumor formation to micrometastasis in ananimal model comprising:a. generating a human prostate cancer xenograftin an immune deficient mouse by implanting intraprostatically locallyadvanced or metastatic prostate cancer tissue, or a cell suspensionthereof from a human into an immune deficient mouse; and b. allowing thexenograft to grow for a time sufficient to permit the detection ofprostate cancer cells within and external to the implant site in theimmune deficient mouse thereby simulating the progression of humanprostate cancer from primary tumor formation to micrometastasis in theanimal model.
 2. The method of claim 1, wherein detection is effected inthe peripheral blood of the immune deficient mouse.
 3. The method ofclaim 1, wherein detection is effected in the bone marrow of the immunedeficient mouse.
 4. A method of stimulating the progression ofosteoblastic bone metastasis in human prostate cancer in a mouse modelcomprising:a. injecting into the tibial bone marrow cavity of an immunedeficient mouse prostate cancer cells prepared from a prostate cancerxenograft generated by implanting into an immune deficient mouse locallyadvanced or metastatic human prostate cancer tissue or a cell suspensionthereof; and b. allowing the injected cells to grow and form anosteoblastic bone lesion which simulates the progression of osteoblasticbone metastasis in human prostate cancer in the mouse model.
 5. An assayfor assessing the effect of a treatment for human prostate cancercomprising:a. applying the treatment to an immune deficient mousebearing a subcutaneous human prostate cancer xenograft generated byimplanting locally advanced or metastatic prostate cancer tissue or acell suspension thereof from a human in the immune deficient mouse; b.determining the effect of the treatment on the growth of the xenograftin said mouse.
 6. An assay for assessing the effect of a gene ofinterest for human prostate cancer comprising:a. introducing the gene toan immune deficient mouse bearing a subcutaneous human prostate cancerxenograft generated by implanting locally advanced or metastaticprostate cancer issue or a cell suspension the immune deficient mouse;b. transducing the cells of the xenograft with the gene in vivo; c.evaluating the presence of micrometastasis in the immune deficient mouseby detecting prostate cancer cells in the peripheral blood, bone marrow,lymph nodes or other sites distant from the site of the subcutaneousxenograft; wherein the effect of the gene on the progression ofmicrometastatic prostate cancer is determined by reference to a controlimmune deficient mouse bearing a subcutaneous human prostate xenograftgenerated with an untransduced subset of the cells of the xenograft. 7.A chimeric mouse model of human prostate cancer, said mouse model havinga human prostate cancer xenograft of locally advanced or metastaticprostate cancer wherein, the mouse model exhibits the transition fromandrogen-dependence to androgen-independence and recapitulates thedevelopment of primary tumors, micrometastasis, and formation ofosteoblastic lesions characteristic of late stage prostate cancer.
 8. Achimeric mouse model of human prostate cancer of claim 7, wherein themouse is a SCID mouse.
 9. A chimeric mouse model of claim 7, wherein thexenograft is implanted subcutaneously.
 10. The mouse of claim 7, whereinthe xenograft is derived from an explant selected from prostate, lymphnode, lung or bone tissue.
 11. A method of producing a chimeric mousemodel of human prostate cancer comprising:a. implanting human prostatecancer tissue designated LAPC-3, -4, or -9 (ATCC deposit Nos. PTA-1440,PTA-1441, and PTA-1442, respectively) into a first imunocompromisedmouse at a site that vascularizes implanted human prostate cancertissue; b. determining whether the human prostate cancer tissue resultsin the mouse model which mimics the clinical transition fromandrogen-dependant to androgen-independent disease; and c. selecting forthe chimeric mouse model that mimics the clinical transition fromandrogen-dependent to androgen-independent disease.
 12. A method ofclaim 11, wherein the imunocompromised mouse is a SCID mouse.
 13. Amethod of claim 11, wherein the human prostate cancer tissue isimplanted into another imunocompromised mouse and the human prostatecancer tissue so implanted maintains human characteristics.
 14. A methodof claim 11, further comprising the steps of:a. harvesting humanprostate cancer tissue from said chimeric mouse model; and b. implantingsaid harvested tissue in a second imunocompromised mouse therebyresulting in chimeric mouse model having passaged human prostate cancertissue.
 15. A method of claim 11, wherein the harvested human prostatecancer tissue is implanted subcutaneously.
 16. The mouse model of claim11, wherein said mouse model mimics the clinical transition fromandrogen-dependent to androgen-independent disease.
 17. A method ofclaim 11, wherein the human prostate cancer tissue is passed to at leastone additional immunocompromised mouse.